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GRIM-19 inhibits v-Src-induced cell motility by interfering with cytoskeletal restructuring.

Sun P, Nallar SC, Kalakonda S, Lindner DJ, Martin SS, Kalvakolanu DV - Oncogene (2009)

Bottom Line: We also show that the N terminus of GRIM-19 played a major role in this process and identified critical residues in this region.More importantly, we show that tumor-associated GRIM-19 mutations disrupted its ability to inhibit v-Src-induced cell motility.These actions appear to occur independently of STAT3, a known target of GRIM-19-mediated inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

ABSTRACT
GRIM-19 (Gene associated with Retinoid-Interferon-induced Mortality 19) is a novel tumor suppressor regulated by interferon/retinoid combination. We have recently shown that GRIM-19 inhibits v-Src-induced oncogenic transformation and metastatic behavior of cells. Oncogenic v-Src induces cell motility by cytoskeletal remodeling, especially the formation of podosomes and. Here, we show that GRIM-19 inhibited the v-Src-induced cell motility by inhibiting cytoskeletal remodeling, that is, podosome formation. We also show that the N terminus of GRIM-19 played a major role in this process and identified critical residues in this region. More importantly, we show that tumor-associated GRIM-19 mutations disrupted its ability to inhibit v-Src-induced cell motility. These actions appear to occur independently of STAT3, a known target of GRIM-19-mediated inhibition. Lastly, tumor-associated GRIM-19 mutants significantly lost their ability to control v-Src-induced metastases in vivo, indicating the biological and pathological significance of these observations.

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Effect of GRIM-19 on v-Src-dependent cell motility(A) Injury-induced migration of cells into the wounded area was monitored. Magnification: 100×. White line indicates the edge of injured site. Note the rapid migration of v-Src-expressing cells, but not the controls, into the wounded area. The wildtype and mutant GRIM-19 proteins were expressed using lentiviral vectors and their impact on cell motility was measured. Note suppression of v-Src-induced cell motility in the presence of wildtype GRIM-19. The mutant proteins have lost such suppressive activity to varying degrees. (B) A quantified view of the inhibitory effects of GRIM-19 on cell motility. Distance migrated from the edge of the monolayer to the center of the denuded area 4h after the induction of injury was calculated from a number of independent samples (n=8) and plotted.
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Figure 5: Effect of GRIM-19 on v-Src-dependent cell motility(A) Injury-induced migration of cells into the wounded area was monitored. Magnification: 100×. White line indicates the edge of injured site. Note the rapid migration of v-Src-expressing cells, but not the controls, into the wounded area. The wildtype and mutant GRIM-19 proteins were expressed using lentiviral vectors and their impact on cell motility was measured. Note suppression of v-Src-induced cell motility in the presence of wildtype GRIM-19. The mutant proteins have lost such suppressive activity to varying degrees. (B) A quantified view of the inhibitory effects of GRIM-19 on cell motility. Distance migrated from the edge of the monolayer to the center of the denuded area 4h after the induction of injury was calculated from a number of independent samples (n=8) and plotted.

Mentions: To determine the biological relevance of the above observations, we performed cell motility assays. Confluent monolayers expressing v-Src, GRIM-19 mutants and their combinations were used for this study. A scratch was induced into these monolayers and the sloughed cells were removed. Cell monolayers were fed with growth medium and the migration of cells into the denuded area was monitored at 4h. Representative data are shown in Fig. 5A. Since the wildtype GRIM-19 or mutants alone were no different from vector control (data not shown), we have only shown the effect of GRIM-19 cell motility in this figure. It is clear from this study that v-Src robustly activates cell motility following the injury, as evidenced by reformation of monolayer. Wildtype GRIM-19 strongly suppressed the v-Src-induced motility. In contrast, the K5N and NΔ17 mutants significantly lost their ability to suppress v-Src-induced cell motility. The R115P mutant was slightly stronger at suppressing v-Src-induced motility than K5N and NΔ17 mutants. Fig.5B shows the quantified view of these data.


GRIM-19 inhibits v-Src-induced cell motility by interfering with cytoskeletal restructuring.

Sun P, Nallar SC, Kalakonda S, Lindner DJ, Martin SS, Kalvakolanu DV - Oncogene (2009)

Effect of GRIM-19 on v-Src-dependent cell motility(A) Injury-induced migration of cells into the wounded area was monitored. Magnification: 100×. White line indicates the edge of injured site. Note the rapid migration of v-Src-expressing cells, but not the controls, into the wounded area. The wildtype and mutant GRIM-19 proteins were expressed using lentiviral vectors and their impact on cell motility was measured. Note suppression of v-Src-induced cell motility in the presence of wildtype GRIM-19. The mutant proteins have lost such suppressive activity to varying degrees. (B) A quantified view of the inhibitory effects of GRIM-19 on cell motility. Distance migrated from the edge of the monolayer to the center of the denuded area 4h after the induction of injury was calculated from a number of independent samples (n=8) and plotted.
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Related In: Results  -  Collection

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Figure 5: Effect of GRIM-19 on v-Src-dependent cell motility(A) Injury-induced migration of cells into the wounded area was monitored. Magnification: 100×. White line indicates the edge of injured site. Note the rapid migration of v-Src-expressing cells, but not the controls, into the wounded area. The wildtype and mutant GRIM-19 proteins were expressed using lentiviral vectors and their impact on cell motility was measured. Note suppression of v-Src-induced cell motility in the presence of wildtype GRIM-19. The mutant proteins have lost such suppressive activity to varying degrees. (B) A quantified view of the inhibitory effects of GRIM-19 on cell motility. Distance migrated from the edge of the monolayer to the center of the denuded area 4h after the induction of injury was calculated from a number of independent samples (n=8) and plotted.
Mentions: To determine the biological relevance of the above observations, we performed cell motility assays. Confluent monolayers expressing v-Src, GRIM-19 mutants and their combinations were used for this study. A scratch was induced into these monolayers and the sloughed cells were removed. Cell monolayers were fed with growth medium and the migration of cells into the denuded area was monitored at 4h. Representative data are shown in Fig. 5A. Since the wildtype GRIM-19 or mutants alone were no different from vector control (data not shown), we have only shown the effect of GRIM-19 cell motility in this figure. It is clear from this study that v-Src robustly activates cell motility following the injury, as evidenced by reformation of monolayer. Wildtype GRIM-19 strongly suppressed the v-Src-induced motility. In contrast, the K5N and NΔ17 mutants significantly lost their ability to suppress v-Src-induced cell motility. The R115P mutant was slightly stronger at suppressing v-Src-induced motility than K5N and NΔ17 mutants. Fig.5B shows the quantified view of these data.

Bottom Line: We also show that the N terminus of GRIM-19 played a major role in this process and identified critical residues in this region.More importantly, we show that tumor-associated GRIM-19 mutations disrupted its ability to inhibit v-Src-induced cell motility.These actions appear to occur independently of STAT3, a known target of GRIM-19-mediated inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

ABSTRACT
GRIM-19 (Gene associated with Retinoid-Interferon-induced Mortality 19) is a novel tumor suppressor regulated by interferon/retinoid combination. We have recently shown that GRIM-19 inhibits v-Src-induced oncogenic transformation and metastatic behavior of cells. Oncogenic v-Src induces cell motility by cytoskeletal remodeling, especially the formation of podosomes and. Here, we show that GRIM-19 inhibited the v-Src-induced cell motility by inhibiting cytoskeletal remodeling, that is, podosome formation. We also show that the N terminus of GRIM-19 played a major role in this process and identified critical residues in this region. More importantly, we show that tumor-associated GRIM-19 mutations disrupted its ability to inhibit v-Src-induced cell motility. These actions appear to occur independently of STAT3, a known target of GRIM-19-mediated inhibition. Lastly, tumor-associated GRIM-19 mutants significantly lost their ability to control v-Src-induced metastases in vivo, indicating the biological and pathological significance of these observations.

Show MeSH
Related in: MedlinePlus